Previous work has demonstrated that several mammalian embryonic cell lines are able to generate functional siRNAs from transfected long dsRNAs and a recent report has demonstrated that embryonic stem cells can also mount a protective antiviral RNAi response. In contrast, terminally differentiated mammalian somatic cells are unable to generate a functionally significant level of siRNAs from either transfected long dsRNAs or from dsRNAs generated during viral infection. We hypothesize that these embryonic cells express a factor required for efficient siRNA production from dsRNAs and we have so far focused on the single mammalian Dicer protein, in part because previous biochemical work has documented the presence of an N- terminal domain that inhibits the ability of human Dicer to process long dsRNAs, but not pre-miRNAs, in vitro. Remarkably, our preliminary data have indeed identified an N-terminally truncated form of Dicer that is expressed in embryonic teratocarcinoma cells but not in human somatic cells. The characterization and functional analysis of this Dicer isoform are key initial goals of this grant application. Once th identity of this novel Dicer isoform is defined, we will express this protein, as well as artificia N-terminal human Dicer deletion mutants and the Drosophila Dcr1/locs-PB and Dcr2/locs-PD protein partners, in a unique human cell line generated in the laboratory, called NoDice, that lacks any functional endogenous Dicer enzyme. We will then determine if we can rescue microRNA processing and reconstitute siRNA production in these cells, in the latter case after challenge with RNA viruses. The main goal of this research is therefore to determine why embryonic mammalian cells are able to mount a protective RNAi response upon viral infection, while somatic cells lack this ability, and to reconstitute RNAi in mammalian cells. We will then analyze whether RNAi can protect cells against viral challenge with a view to later extending this research into in vivo models of viral pathogenesis. Overall, this project has the potential to not only reveal why RNAi is non- functional in mammalian somatic cells but also to allow the effective reconstitution of this potentially highly protective antiviral innate immune response, initially in cultured cells but potentially later also in vivo.